JPH09257797A - Article for immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simplify the operation of immunoassay and to dispense with a strict condition by fluorinating the surface of a region bearing immunoeaction by discharge plasma treatment to enhance the adsorbing characteristics thereof. SOLUTION: For example, beads made of polystyrene are subjected to discharge plasma treatment under pressure near to atmospheric pressure within an apparatus wherein internal and external electrodes 3, 4 are arranged in a treatment container 1 made of a dielectric such as glass and an AC power supply 2 is provided. Beads are continuously supplied from above and mixed gas obtained by diluting F-containing gas such as C3 F6 by inert gas such as He is supplied from below. For example, if voltage of 15kHz and 10kV is applied across electrodes to generate discharge plasm, beads of which the entire surfaces are subjected to discharge plasma treatment are obtained and the F/C ratio of the surfaces of them is about 2. These beads are high and uniform in the adsorbing property of an antibody as compared with untreated beads. Even with respect to microtiter plate, the same result can be obtained by a method similar to the above mentioned method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoassay instrument which can be suitably used for immunoassay.

[0002]

2. Description of the Related Art Conventionally, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (EI) have been used as highly sensitive immunoassays.
Various methods such as A, ELISA) and fluorescence immunoassay (FIA) are known. In recent years, research on automation of those measurements has been advanced, and some automatic analyzers have been put on the market, especially in EIA.

Such an automatic analyzer uses a solid phase method for B / F separation. The solid phase method is a method in which an antigen or antibody is bound to a carrier such as a plastic plate, a plastic piece, plastic beads, glass beads, or a tube, and an antigen-antibody reaction and an unreacted antigen or antibody are used. It is a method of separating and by a simple operation.

Examples of the material of the beads used as the carrier in these automatic analyzers include glass and the like.
A automatic analyzer (PK-300, Olympus Industrial Co., Ltd.)
There was an example of using glass beads. However, glass beads are fragile, it is difficult to make one with a certain size, and it takes time and effort to bind the antibody or antigen to the beads. There was a problem that it was difficult to make a good product.

On the other hand, in order to make up for the drawbacks of glass beads, RIA and EIA kits using polystyrene, ABS or other plastic beads as the solid phase have been released by various companies. The plastic beads do not have the above-mentioned problems such as fragility and difficulty in producing a certain size. Among them, polystyrene beads are one of the most suitable for immobilizing antibodies and antigens and are widely used.

Japanese Unexamined Patent Publication No. 1-131460 discloses a technique using immunoassay beads made of a fluorine-containing resin having a specific gravity in the range of 1.4 to 2.5. Is difficult to mold, and it is necessary to operate it under a skilled technique, that is, strictly controlled temperature and time control for the adsorption operation of the antigen or antibody.

Japanese Unexamined Patent Publication (Kokai) No. 62-123359 discloses a microtiter plate used as the above carrier in an automatic analyzer after low-pressure plasma treatment of the plate surface,
A molded article and a method for producing the same are disclosed, in which the antigen-antibody is easily adsorbed by treating with a peroxide. But,
This method also requires a skillful technique for adsorption operation of the antigen or antibody, that is, operation under strictly controlled temperature and time control.

[0008]

SUMMARY OF THE INVENTION In view of the above, an object of the present invention is to provide an immunoassay instrument which is easy to operate and does not require operation under strict conditions.

[0009]

DISCLOSURE OF THE INVENTION The present invention is an immunoassay product having an immune reaction site, wherein at least the surface of the immune reaction site is a discharge plasma generated in the presence of a fluorine-containing gas. It is an immunoassay device that is fluorinated by contacting with. Hereinafter, the present invention will be described in detail.

The immunoassay product of the present invention has a site responsible for an immune reaction. Examples of the immunoassay product include beads and microtiter plates. In the present invention, the site responsible for the immunological reaction is a site where the immunological reaction is actually carried out in the immunoassay using the above-mentioned immunoassay product, and when the immunoassay product of the present invention is beads. The site responsible for the immune reaction means the entire surface of the beads, and when the immunoassay device of the present invention is a microtiter plate, the site responsible for the immune reaction is the inner surface of the well of the microtiter plate. Means

When the immunoassay article of the present invention is beads, the material of the beads is not particularly limited, but in view of moldability, a composition containing a thermoplastic resin as a main component is preferable. In general, polystyrene, ABS, polypropylene and the like are used as the material for the immunoassay beads because of their excellent ability to adsorb antibodies and antigens, but these are also preferably used in the present invention. The beads preferably have a specific gravity in the range of 1.03 to 2.5 as a whole of the composition in consideration of the use form when applied to an automatic analyzer, analysis accuracy, and the like. It may be composed of only a resin, or may be a mixture of a resin having a relatively high specific gravity and a resin having a low specific gravity. Resins such as polystyrene that are widely used as beads for immunoassay are excellent in the ability to adsorb antibodies and antigens, but since their specific gravity is lower than the above range, resins with high specific gravity,
It is possible to apply the present invention by adding a filler or the like to adjust the specific gravity of the entire composition within the above range.

The resin having a high specific gravity which can be used for adjusting the specific gravity as the material for the beads is, for example, monofluoroethylene, difluoroethylene, trifluoroethylene, or 4
Examples thereof include halogen-containing resins containing a monomer such as ethylene fluoride, vinylidene fluoride, vinylidene difluoride, vinylidene trifluoride, vinylidene fluoride, and vinylidene fluoride as a polymerization component.

As the above-mentioned filler, one having a relatively large specific gravity (about 2 to 5) and capable of increasing the apparent specific gravity of the beads per se with a small amount and having no influence on the antigen-antibody reaction is used. . Examples of such a material include barium sulfate powder, calcium carbonate powder, glass powder, and glass fiber. Among them, barium sulfate powder is preferable. These may be used as a mixture of two or more if necessary.

The above-mentioned filler is contained in an amount of 20 parts by weight or less, preferably 5 to 15 parts by weight, based on 100 parts by weight of the thermoplastic resin, and the specific gravity of the entire composition is adjusted to a range of 1.03 to 2.5. To be done. The above composition is molded into a spherical shape by a conventional method such as injection molding or extrusion molding. The beads have a diameter of 1-10 mm, preferably 3-10 mm.

When the immunoassay product of the present invention is a microtiter plate, the material of the microtiter plate is not particularly limited, but in view of moldability, a composition containing a thermoplastic resin as a main component is preferable. .
In general, as materials for microtiter plates for immunoassay, polymethacrylate (PMMA), polystyrene,
Although ABS, polypropylene and the like are used, it is preferable to use them also in the present invention. The above material is molded into a microtiter plate having wells standardized by a conventional injection molding method.

In the present invention, the surface of the site responsible for the above-mentioned immune reaction is fluorinated by contacting discharge plasma generated in the presence of a fluorine-containing gas. The fluorine-containing gas is not particularly limited, and examples thereof include carbon tetrafluoride (CF ₄ ) and carbon hexafluoride (C ₂ F
₆ ), Fluorohydrocarbon gas such as propylene hexafluoride (C ₃ F ₆ ) and octafluorocyclobutane (C ₄ F ₈ ); Halogenated hydrocarbon gas such as monochlorotrifluorocarbon (CClF ₃ ); Examples thereof include sulfur fluoride compounds such as sulfur hexafluoride (SF ₆ ). Among them, carbon tetrafluoride, carbon hexafluoride, propylene hexafluoride, cyclobutane octafluoride and the like are preferably used because they are safe and do not generate harmful gases such as hydrogen fluoride. In particular, a fluorocarbon gas having an unsaturated bond such as propylene hexafluoride or octafluorocyclobutane, which tends to increase the amount of fluorination, is more preferably used.

The discharge plasma treatment can be carried out by supplying a fluorine-containing gas by the usual plasma treatment method. In the present invention, the discharge plasma treatment is not particularly limited as long as the surface of the immune reaction molded article can be fluorinated. Generally, discharge plasma treatment is performed under low pressure conditions close to vacuum, but according to the treatment apparatus described in the embodiment of the present invention, it is possible to perform treatment under a pressure near atmospheric pressure,
It is advantageous in terms of the simplicity of the apparatus, the ease of continuous processing, the economy, and the like. The pressure under the atmospheric pressure means 100 to 80.
It is under a pressure of 0 Torr, and the range of 700 to 780 Torr is preferable because the pressure can be easily adjusted and the operation is simple.

When the discharge plasma treatment is performed under a pressure near atmospheric pressure, the fluorine-containing gas is used after being diluted with an inert gas. Examples of the above-mentioned inert gas include helium, neon, argon, xenon, etc. These rare gases may be used alone or in combination. However, since the metastable state has a long life, it is preferable to use helium, which is advantageous for exciting the processing gas. When an inert gas other than helium is used, it is preferable to mix and use an organic compound gas such as acetone, methanol, methane, or ethane at 2% by volume or less.

The dilution ratio between the fluorine-containing gas and the inert gas is appropriately determined according to the type of gas used.
It is necessary that the concentration of the fluorine-containing gas is 0.1 to 10% by volume or less, and a range of 0.1 to 5% by volume is more preferable. When the concentration of the fluorine-containing gas exceeds 10% by volume,
Even if a high voltage is applied, discharge plasma is unlikely to be generated, and it may shift to arc discharge, so that good surface fluorination treatment cannot be performed.

The discharge plasma is generated by applying an AC voltage having a frequency on the order of kHz.
A low frequency of 0 kHz is particularly preferred. The voltage at that time is appropriately determined, but it is preferable to apply the voltage to the electrode and apply the voltage so that the electric field strength thereof is about 1 to 40 kV / cm. When the electric field strength is less than 1 kV / cm,
The treatment takes time and is inefficient, and conversely, when the electric field strength exceeds 40 kV / cm, the behavior of transition to arc discharge is exhibited.

The immunoassay product of the present invention can be used in various immunoassay methods. As the antigen to be measured,
There are various biological components or drugs, and clinically, various hormones and proteins contained in biological fluid are clinically important. Examples thereof include peptide hormones; thyroid hormones such as thyroxine; cancer markers such as immunoglobulin, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), and nerve-specific enolase (NSE). The antibodies measured include antibodies produced in vivo by various infectious diseases, antibodies produced by autoimmune diseases, antibodies produced against various drugs, and the like.

The adsorption operation is carried out by immersing the beads of the present invention used as a carrier in a buffer solution containing the antigen or antibody, or by putting the buffer solution containing the antigen or antibody into the reaction well of the microtiter plate of the present invention. To do. The adsorption conditions are 0 to 50 ° C. and 1 to 48 hours, preferably 4 to 4
It is 0 degreeC and 2 to 6 hours. However, when the immunoassay product of the present invention is used, strict temperature and time control as in conventional products is not required. The buffer solution is not particularly limited as long as it can be used for adsorption of an antigen or an antibody, and is preferably a phosphate buffer solution or a borate buffer solution.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION An example of discharge plasma processing will be described below with reference to the drawings. 1 and 2 are schematic cross-sectional views of a discharge plasma processing method for beads. This device is composed of a discharge treatment container 1, an AC power supply 2, an internal electrode 3, and an external electrode 4. The beads are put into the discharge treatment container 1 adjusted to the gas atmosphere and subjected to discharge plasma treatment. An internal electrode 3 is arranged in the center of the discharge treatment container 1, and an external electrode 4 is arranged on the outer surface of the container 1. As the electrode arrangement, a known capacitive coupling type or inductive type is used.

The discharge processing container 1 is made of a solid dielectric material. Examples of the solid dielectric include polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), polymethacrylate (PMM).
A), made of plastic such as polycarbonate (PC);
Made of glass such as borosilicate glass, soda glass, and quartz;
Examples thereof include metal oxides such as aluminum oxide (Al ₂ O ₃ ), zirconium oxide (ZrO ₂ ), titanium dioxide (TiO ₂ ), and mixtures thereof.

The electrodes 3 and 4 may be made of a multi-component metal such as stainless steel or brass, or may be made of a pure metal such as copper or aluminum. When the electrodes 3 and 4 directly face each other, the discharge state shifts to arc discharge. Therefore, in this device, the discharge treatment container 1 made of a solid dielectric is used.
The external electrode 4 is attached to the outer surface of the electrode so that the solid dielectric is always positioned between the electrodes 3 and 4. On the contrary, it is also possible to adopt a mode in which the above-mentioned solid dielectric is arranged inside a container having a metal type cylindrical wall which also serves as an electrode.

If the solid dielectric is too thick, a high voltage is required to generate discharge plasma, and if it is too thin, dielectric breakdown occurs when a voltage is applied and arc discharge easily occurs. A thickness of 4 mm is preferred.

When the beads are brought into contact with the discharge plasma generated by applying a voltage between the electrodes 3 and 4, the bead surface is fluorinated. The above-mentioned fluorination treatment may be carried out by heating or cooling the substrate, but it is sufficiently possible at room temperature. The time required for the fluorination treatment is appropriately determined depending on the input power, the gas type, the gas composition, and the like. The amount of fluorination on the surface of the beads is appropriately determined depending on the antigen, but fluorine / carbon (atomic ratio) = 0.5 to 3 is preferable, and a larger value tends to be more preferable.

FIG. 3 is a schematic cross-sectional view of the discharge plasma treatment method for the inner surface of the well of the microtiter plate. This device includes an AC power supply 11, a working electrode 12, and a counter electrode 13.
It is composed of The AC power supply 11 can generate a voltage having a frequency on the order of kHz, but a low frequency of 5 to 30 kHz is preferable when processing a substrate having low heat resistance. The generation of the discharge plasma is performed by applying the above-mentioned AC voltage, but it is possible to apply a DC voltage of positive and negative polarities by weight, if necessary, and the applied voltage and the polarity depend on the gas composition used. It is decided as appropriate.

The working electrode 1 is provided inside the well of the plate 14.
2, the counter electrode 13 is arranged on the bottom of the outer surface, and the electrode 1
By applying an AC electric field between 2 and 13, discharge plasma is generated in the plasma generation part 5 which is between the working electrode 12 arranged inside the container and the counter electrode 13, and the discharge plasma is generated. The inner bottom portion of the well contacting with is fluorinated.

The working electrode 12 also serves as a conduit that passes through the well opening for supplying the mixed gas into the well and opens inside the well. Working electrode 12 and counter electrode 1
3 (the shortest distance between the electrodes in FIG. 3) is appropriately determined depending on the thickness of the plate 14, the magnitude of the applied voltage, the gas composition, etc., but is preferably between 0.1 and 30 mm. If it exceeds 30 mm, the uniformity of the discharge plasma tends to be impaired. Examples of the metal used as the electrode include multi-component metals such as stainless steel and brass;
Examples include pure metals such as copper and aluminum. Although not shown, the flow rate of the mixed gas is controlled by a mass flow controller.

[0031]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Examples 1 to 6 Preparation of beads Using the composition shown in Table 1, spherical beads having a diameter of 6.7 mm were prepared by a known injection molding method, and 100 beads each were prepared as a physicochemical cleaning agent (SCAT20XN). (Manufactured by Hanai Chemical Co., Ltd.) and washed with an ultrasonic cleaner for 10 minutes. Then, after thoroughly washing with purified water, it was air-dried to obtain beads 1, 2 and 3. However, for the beads 2, barium sulfate powder was added to polystyrene 10.
The content was 10 parts by weight with respect to 0 parts by weight.

[0033]

[Table 1]

The beads thus obtained were subjected to discharge plasma treatment using the apparatus shown in FIG. In the above apparatus, a borosilicate glass tube having an outer diameter of 20 mm (inner diameter of 14 mm) x a length of 1000 mm is used as a processing vessel, a copper wire electrode having a diameter of 2 mm x a length of 1000 mm is in contact with the outside of the vessel, and a diameter of 20 mm is a length of 1000 mm.
It is an apparatus in which a copper tube electrode of mm is arranged, and is inclined by about 20 degrees. The beads were continuously supplied from above, and the diluted mixed gas of the fluorine-containing gas and the inert gas was supplied from below the device. By applying a voltage of 15 kHz between the electrodes and the magnitude shown in Table 2, discharge plasma is generated inside the glass tube, and the discharge plasma is supplied to the inside of the glass tube and comes into contact with the beads rolling down the slope. Then, beads whose entire surface was subjected to discharge plasma treatment were obtained. When the discharge plasma treatment was performed under atmospheric pressure conditions with the mixing ratio of the fluorine-containing gas being 15% by volume of the whole, arc discharge was confirmed. The quantification of the fluorination amount of the obtained beads and the adsorption characteristic test were conducted by the following methods, and the results are shown in Table 2.
It was shown to.

Quantification of Fluorination Amount The amount of surface fluorine of the beads treated with the discharge plasma is X.
It was measured by line electron spectroscopy. Table 2 shows the atomic ratio of fluorine / carbon.
It was shown to. The surface of the beads of the example has an atomic ratio of 1.5-2.
It was confirmed that it was fluorinated in 2. The atomic ratio of the treated product in the atmosphere of the fluorine-containing gas in which the arc discharge was generated was 15% was 0.3.

Adsorption Characteristic Test Ten beads under each condition were prepared for performance evaluation. Put one bead of each condition into a 10 ml test tube,
Next, add 1 ml of mouse IgG solution, soak it at 4 ° C for 24 hours, discard the solution, wash with a washing solution, and add 1% BS.
3 ml of solution A was added and blocking was performed at room temperature for 1 hour. Then, peroxidase-labeled anti-mouse IgG
After adding 1 ml of the solution and then washing, a coloring solution was added, the mixture was reacted at room temperature for 15 minutes, and a stop solution was added. 1m of colored liquid
Samples were taken one by one and the absorbance at 492 nm was measured.

The above reagents were prepared as follows. Mouse IgG solution : Mouse IgG (manufactured by Seikagaku Corporation) in 0.1 M phosphate buffer (pH 6.5) at 100 μg / m
It was dissolved so that it became l. 1% BSA solution : 0.1M containing 1% bovine serum albumin
Phosphate buffer (pH 6.5). Wash solution : physiological saline containing 0.05% Tween 20. Peroxidase-labeled anti-mouse IgG solution : 1% B of peroxidase-labeled anti-mouse IgG (manufactured by Seikagaku Corporation)
Diluted 2000 times with SA solution. Color developing solution and reaction stopping solution : Kit manufactured by Towns (Substrate: O
PD) is used.

Comparative Examples 1 to 3 The adsorption characteristics test was conducted on the polystyrene beads 1, 2 and 3 used in the examples without discharging plasma. The results are shown in Table 2.

[0039]

[Table 2]

Examples 7 to 12 A discharge plasma treatment was performed on a polystyrene microfabricated microtiter plate (manufactured by Nunc) with the apparatus shown in FIG. The working electrode 12 of the above apparatus was made of stainless steel and provided with 4 × 8 conduits having an outer diameter of 5 mm, an inner diameter of 3 mm and a height of 7 mm at intervals of 9 mm. The diluted mixed gas of the fluorine-containing gas and the inert gas was supplied into the cell through the conduit of the electrode 12. The counter electrode 13 is 80 × 40 × 1 m
m copper plate was used. Processing is performed in 4 rows (32 pieces) at the same time,
One row (8 pieces) was used for contact angle measurement, one row was for quantitative determination of the amount of fluorine, and the other two rows were for adsorption characteristic test. 1 between electrodes 2 and 3
By applying a voltage of 5 kHz and a magnitude shown in Table 3, discharge plasma was generated inside the well to perform surface treatment.
When the discharge plasma treatment was performed under atmospheric pressure conditions with the mixing ratio of the fluorine-containing gas being 15% of the whole, arc discharge was confirmed. Contact angle measurement of the obtained well,
The quantification of the amount of fluorination and the adsorption characteristic test were conducted by the following methods. The results are shown in Table 3.

Contact angle measurement: The bottom of the well was cut out, and 2 μl of water droplet was dropped, and a contact angle measuring device (trade name CA-X150, manufactured by Kyowa Interface Science Co., Ltd.) was used.
Was used to measure the static contact angle. Quantification of fluorination amount It was measured in the same manner as in Example 1. It was confirmed that the treated product of the example had an atomic ratio of 1.0 to 1.5 and the inner surface of the well was fluorinated. The atomic ratio of the treated product in the atmosphere of 15% fluorine-containing gas in which the arc discharge occurred was 0.3.

Adsorption characteristic test An adsorption characteristic test was carried out on a 96-well plate as follows. 200 μl of a mouse IgG solution was added to each well, and the plate well was allowed to stand overnight at 4 ° C. to cause an adsorption reaction to the plate well. Then, the solution was discarded, washed with a washing solution, 300 μl of a 1% BSA solution was added, and blocking was performed at room temperature for 1 hour. Then, wash and add 200 μl of peroxidase-labeled anti-mouse IgG solution, and after washing, add a coloring solution and let the enzyme-substrate reaction proceed at room temperature for 15 minutes, then add a reaction stop solution to stop the reaction, and absorb at 492 nm. Was measured with an EIA plate reader. The reagent is
It adjusted like Example 1.

Comparative Example 4 An adsorption characteristic test was conducted on the polystyrene plate of Example 7 without applying discharge plasma. The results are shown in Table 3.

[0044]

[Table 3]

[0045]

INDUSTRIAL APPLICABILITY The immunoassay product of the present invention is easy to operate, does not require strict conditions, and can adsorb many antibodies.
The amount of adsorption is made uniform. Therefore, it contributes to reduction of the amount of antibody used, shortening of antigen-antibody reaction time, and improvement of measurement accuracy.

[Brief description of drawings]

FIG. 1 is an explanatory view of an apparatus for producing beads of the present invention.

FIG. 2 is a cross-sectional view of an apparatus for making beads of the present invention.

FIG. 3 is an illustration of an apparatus for making the plate of the present invention.

[Explanation of symbols]

 1 Processing Container (Glass Tube) 2 AC Power Supply 3 Internal Electrode 4 External Electrode 11 AC Power Supply 12 Working Electrode 13 Counter Electrode 14 Plate

Claims (5)

[Claims] 1. An immunoassay device having an immune reaction site, wherein at least the surface of the immune reaction site is fluorinated by contacting discharge plasma generated in the presence of a fluorine-containing gas. An immunoassay product characterized by being a product. 2. The immunoassay article is a bead, and the surface of the site responsible for the immune reaction is the entire surface of the bead.
The immunoassay product described. 3. The immunoassay instrument according to claim 1, wherein the immunoassay instrument is a microtiter plate, and the surface of the site responsible for the immune reaction is the inner surface of the well of the microtiter plate. 4. The immunoassay instrument according to claim 1, wherein the fluorine-containing gas contains a fluorocarbon gas having an unsaturated bond. 5. The immunoassay instrument according to claim 1, wherein the discharge plasma is generated under a pressure near atmospheric pressure.